Macromolecular complexes are of key importance in virtually all life processes. However, more powerful methods need to be developed to (1) validate potential interacting partners, (2) efficiently screen for complex formation, and (3) produce sufficient amounts of functional protein complexes for in depth functional and structural studies. To address these needs we will (1) develop new high-throughput fluorescence-based screens to assess protein-protein interactions;(2) develop rapid screening method to distinguish between transient and stably interacting partners. (3) Finally, since functional characterization and structure determination of protein complexes depends on the capability to produce them recombinantly in high yield, we will develop a multi-protein expression system, based on distinct fluorescent markers whose expression levels are correlated with each protein component. Together, these methods provide a high- throughput pipeline for validating, characterizing, and producing protein complexes for structural and functional studies. This platform, and the associated reagents developed under this proposal, will provide a much needed toolbox that will greatly aid any scientist studying eukaryotic protein complexes. PUBLIC HEALTH RELEVANCE: Multi-protein complexes are of key importance to virtually all life processes, but difficulties in identifying the components of these often-intricate assemblies, and producing them in vitro, have hampered progress. We propose new methods for identifying and producing multi-protein complexes based on the use of fluorescent markers. This new technology should enable heretofore intractable studies of protein complexes, and thus have wide-ranging impact on medical science.